Gene name - hibris
Cytological map position - 51D11--E1
Function - receptor
Keywords - muscle fusion
Symbol - hbs
FlyBase ID: FBgn0029082
Genetic map position -
Classification - nine Ig-C2 type repeats and a Fibronectin type III domain
Cellular location - surface transmembrane
Hibris (Hbs) is a transmembrane immunoglobulin-like protein that shows extensive homology to Drosophila Sticks and stones (Sns) and the human kidney protein Nephrin. Hbs is expressed in embryonic visceral, somatic and pharyngeal mesoderm among other tissues. In the somatic mesoderm, Hbs is restricted to fusion competent myoblasts (the cells that fuse to founder cells) and is regulated by Notch and Ras signaling pathways. Embryos that lack or overexpress hbs show a partial block of myoblast fusion, followed by abnormal muscle morphogenesis. Abnormalities in visceral mesoderm are also observed. In vivo mapping of functional domains suggests that the intracellular domain mediates Hbs activity. Hbs and its paralog, Sns, co-localize at the cell membrane of fusion-competent myoblasts. The two proteins act antagonistically: loss of sns dominantly suppresses the hbs myoblast fusion and visceral mesoderm phenotypes, and enhances Hbs overexpression phenotypes. hbs is not continuously expressed in all fusion-competent myoblasts during the fusion process. S2 cell aggregation assays have revealed a heterotypic interaction between Hibris and Kin-of-irre (Kirre, formerly Dumbfounded), but not between Hibris and Irregular Chiasm-Roughest (Dworak, 2001). It is proposed that Hibris is an extracellular partner for Dumbfounded and potentially mediates the response of myoblasts to this attractant. The temporal pattern of hbs expression within fusion-competent myoblasts may reflect previously undescribed functional differences within this myoblast population (Artero, 2001; Dworak, 2001).
The general framework for understanding muscle cell fusion was provided by Doberstein (1997), who subdivided the process into steps: cell-cell recognition, adhesion, alignment of membranes and membrane fusion. Genetic studies in Drosophila have also provided fusion mutants (Paululat, 1999). Essential loci for myoblast fusion include the transcriptional regulator Mef2, the membrane bound protein Rolling stone (Paululat, 1997), cytoplasmic proteins such as Blown fuse (Doberstein, 1997), and components of the Rac1 signaling pathway such as Myoblast city and Drac1. Interestingly, overexpression of weak gain-of-function Notch constructs throughout the mesoderm can completely block fusion without interfering with earlier roles for Notch, suggesting that Notch is also involved in muscle morphogenesis. Another family of proteins that plays crucial roles in myoblast fusion in Drosophila is the immunoglobulin (Ig) superfamily. These Ig-containing proteins include Kirre (Ruiz-Gómez, 2000) and Sns (Bour, 2000). Although the described mutant phenotypes for kirre and sns are the same -- a complete fusion block -- their expression patterns in the somatic mesoderm are strikingly different. kirre is expressed exclusively in founder cells, while Sns is expressed exclusively in fusion-competent cells. Moreover, kirre is expressed during the entire fusion process and acts as an attractant for myoblasts, indicating that founder cells actively recruit fusion-competent cells until the final muscle size is achieved (Ruiz-Gómez, 2000). Sns, however, is a general marker for fusion-competent cells and, consistent with the segregation of these myoblasts, its expression depends on Notch signaling (Bour, 2000). Both Kirre and Sns are involved in the initial steps of myoblast cell-cell recognition, since free myoblasts in embryos mutant for either gene do not cluster around founder cells, suggesting that there is no recognition between founder cells and fusion-competent cells (Artero, 2001 and references therein).
To better understand muscle diversity and morphogenesis, a differential display screen was devised to identify genes specifically expressed in founder versus fusion-competent myoblasts. In short, use was made of the observation that muscle progenitor specification depends on Notch and Ras signaling to either repress or enhance muscle progenitor fate, respectively. Activated forms of Notch and Ras were overexpressed in Toll10b mutant embryos to reduce tissue complexity. Toll10b mutant embryos differentiate almost exclusively as somatic mesoderm (Artero, 2001).
A single differentially displayed band was chosen for further study because it was strongly upregulated under the activated Notch conditions. Corresponding cDNAs were isolated and the gene was named hibris. hbs is indeed dependent on Notch signaling. Northern blot signals suggested that hbs expression is upregulated at least twofold upon Notch activation, and repressed at least fivefold upon Ras activation (Artero, 2001).
Although hbs mutant flies survive to adult stages in normal numbers and show visible phenotypes such as rough eyes, reduced viability and semisterility, clear abnormalities are found in muscle development in mutant embryos. Embryos heterozygous for hbs459/Df(2R)X28, and different hbs transallelic combinations, were probed with anti Myosin antibody and compared with their wild-type counterparts. In embryos lacking hbs function, it was found that the muscle pattern is specified, although some muscles are occasionally missing or show abnormal morphologies (e.g. look thinner than normal or have fewer nuclei than normal). Immunocytochemistry with antibodies directed against founder cell markers such as Krüppel, Even-skipped and Runt reveals a normal pattern. These embryos, however, reproducibly showed a partial fusion block: an increased number of free myoblasts are present when compared with wild-type embryos. Unfused myoblasts are detected scattered around the muscles, surrounding the gut, heart and CNS. These free myoblasts show extended processes, indicative of their competence to scan for founder cells. In these experiments, stage 16 embryos were studied to ensure that free myoblasts have had the chance to fuse and that in this mutant background they fail to do so. Note, however, that by this developmental stage many unfused myoblasts might have undergone apoptosis, been phagocytosed and lost Myosin expression, decreasing the apparent number of unfused myoblasts. Loss of hbs function also interferes with the normal gut development as shown both by an enlargement of the first chamber as well as by loss of visceral muscle progenitors at stage 12 (Artero, 2001).
The GAL4/UAS system was used to assess the effect of hbs overexpression. When UAS-hbsFL was expressed throughout the embryonic mesoderm driven by twist-GAL4;DMef2-GAL4, defects were detected both in the somatic and visceral mesoderm similar to loss of hbs function. In the somatic mesoderm, a partial fusion block with free myoblasts scattered at several locations was found. In addition, these embryos normally show a greater degree of muscle loss than hbs homozygous mutant embryos. Defects in the visceral mesoderm, as revealed by anti-Fasciclin III staining, were also found. Because delivering extra Hbs to both founder and fusion-competent myoblasts leads to muscle losses, tests were performed to see whether all founder cells were correctly specified in these embryos. Antibody staining with the founder cell markers Krüppel, Even-skipped and Runt indicated that founder cells are correctly specified in embryos overexpressing Hbs. However, at later stages, aberrant morphologies were detected in the growing muscles (i.e. odd shapes), suggesting that prolonged Hbs expression interferes with muscle morphogenesis rather than the initial specification (Artero, 2001).
To dissect which parts of the protein are important for Hbs function, two additional constructs were tested. The constructs either deleted the extracellular (UAS-hbsDeltaECD) or the intracellular (UAS-hbsDeltaICD) region of the protein, but maintained the transmembrane domain such that the truncated proteins remain membrane anchored. Overexpression of UAS-hbsDeltaECD in the mesoderm results in detectable phenotypes, indicating that the intracellular domain of Hbs alone can interfere with muscle development. Specifically, a greater degree of fusion block and muscle loss as well as defects in the late pattern of founder cell markers were detected compared with overexpression of the full-length Hbs. This construct also causes greater defects in visceral mesoderm, leading to a greater reduction of visceral muscle progenitors. Altogether, these results suggest that the cytoplasmic domain of Hbs is required for hbs function in the mesoderm. By contrast, overexpression of UAS-hbsDeltaICD does not affect somatic muscle development appreciably but blocks visceral mesoderm development (Artero, 2001).
Since Sns, Kirre and Roughest are all Ig-C2-containing proteins implicated in myoblast fusion, their possible functional relationship with hbs was examined by testing for genetic interactions. For this analysis Df(1)w67k30 was tested: this deficiency removes both kirre and roughest, both of which are suggested to be partially redundant in the fusion process. hbs and Df(1)w67k30 do not interact genetically, either in loss- or gain-of-function hbs backgrounds. In addition, overexpression of hbs does not rescue any aspect of the Df(1)w67k30 phenotype (Artero, 2001).
Hbs and Sns co-localize at the cell membrane at discrete points, which would be consistent with both proteins working together in the fusion process. The doses of sns and hbs were manipulated. Stage 16 hbs homozygous mutant embryos characteristically show groups of free myoblasts throughout the embryo, and a gut phenotype was revealed early by anti-Fasciclin III or late by anti-Myosin staining. hbs-null embryos in which the sns dose is halved show dominant suppression of both the partial fusion block and gut phenotype. When hbs and sns double mutant embryos were analyzed, no noticeable change in the sns phenotype was detected. Thus, sns and hbs appeared to act antagonistically (Artero, 2001).
Consistent with the hypothesis that hbs and sns act antagonistically rather than cooperatively in myoblast fusion, sns mutations dominantly enhance hbs overexpression phenotypes. In the somatic muscles, there is an increase in the number of free myoblasts. This interaction also leads to a greater loss in visceral muscle progenitors, as measured by Fasciclin III expression. To quantify these observations, the number of gaps in Fasciclin III expression in stage 12 embryos was used as a measurement of genetic interaction between hbs and sns. The result of this quantification is consistent with the qualitative analysis. The dominant increase in Fasciclin III expression gaps when sns dose is lowered is statistically significant at the P<0.0001 level, indicating that a synergistic, rather than an additive, effect between hbs overexpression and heterozygosity for sns accounts for the increase in Fasciclin III expression gaps observed. As an additional test to determine the relationship between hbs and sns, attempts were made to rescue the sns loss-of-function phenotype by overexpression of hbs. In this experiment, Hbs does not rescue the sns loss-of-function phenotype in the somatic muscles in agreement with the genetic data. Altogether, the results of these experiments suggest that hbs antagonizes sns function during mesoderm development, but cannot differentiate whether sns is downstream of hbs or both genes are working in parallel pathways (Artero, 2001).
These data show that hbs overexpression gives a similar phenotype to hbs loss and suggest that either Hbs transduces a signal or these constructs behave as dominant negatives. In support of Hbs acting as a signal transducer, it was found that overexpression of UAS-hbsDeltaECD results in a partial block in somatic muscle development, indicating that the cytoplasmic domain of Hbs mediates the activity of the protein. A similar dependence on the cytoplasmic domain of Notch in the embryonic nervous system or EGF receptor signaling pathway antagonist Echinoid in the eye imaginal discs (Bai, 2001) has been used as support for these proteins acting as signal transducers. Alternatively, the overexpression data with both the full-length and intracellular domain constructs could be interpreted as Hbs exerting a dominant negative effect through the titration of another crucial component, such as another membrane protein or cytoskeletal component. This key protein could be a target of Hbs without Hbs actually transducing a signal in the classical sense. However, since the behavior of mutant combinations of sns and hbs is different (Hbs loss of function is suppressed by sns mutations whereas hbs overexpression is enhanced by sns mutations) the possibility is favored that Hbs transduces a signal rather than behaves as a dominant negative in the overexpression studies. It would be expected that sns heterozygosity would suppress both the hbs loss and overexpression phenotypes if hbs overexpression were acting as a dominant negative. These data also indicate that the extracellular domain of hbs does not behave as a dominant negative construct either, at least not in the somatic mesoderm, because overexpression of this construct does not cause a detectable phenotype in the somatic musculature (Artero, 2001).
Given the lack of enzymatic features in the cytoplasmic domain, it is proposed that Hbs associates with adapter proteins to connect to the cytoskeleton, cytoplasmic kinases or other transmembrane receptors. Interestingly, the intracellular domain contains motifs that are conserved in Sns. These motifs could potentially serve as interfaces for adaptor proteins. Work with Nephrin provides a putative candidate, the mouse CD2-associated protein (CD2AP). CD2AP, an SH3-containing cytoplasmic protein, has been shown to interact with the intracellular domain of Nephrin and has been proposed to anchor Nephrin to the actin cytoskeleton at the slit diaphragm in the kidney (Shih, 1999) and to participate in setting up the immunological synapse in T cells (Dustin, 1998). The Drosophila genome contains a CD2AP homolog, which is currently being analyzed (Artero, 2001).
Overexpression of UAS-hbsDeltaECD or UAS-hbsFL throughout the mesoderm results in a partial fusion block, but with greater muscle loss and morphological defects when compared with the loss of function phenotype. This increased muscle loss could reflect that, in overexpression experiments, Hbs is expressed in both fusion-competent as well as founder cells, thereby breaking the expression asymmetry that is found under normal situations. However, it could equally be supposed that prolonged and higher levels of Hbs block muscle fusion and disrupt later events in muscle morphogenesis. It is noted that both loss and gain of hbs result in a fusion phenotype. Although it is obvious why gain of a negative regulator could result in a fusion block, it is less clear why loss results in a similar phenotype. It is speculated that hbs highlights a novel aspect of the regulated events in the fusion process (Artero, 2001).
Cell recognition and adhesion between myoblasts is the first step of the fusion process (Doberstein, 1997). Kirre has been shown to be expressed in founder cells and behave as a myoblast attractant. By contrast, Sns is expressed in fusion competent myoblasts exclusively. A simple model can be proposed with these data: Kirre interacts at the cell surface with Sns to allow recognition between founder and fusion-competent cells (Taylor, 2000; Frasch, 2000). Subsequently, this recognition would signal the start of an orderly fusion process that requires such proteins as Rac, Blown fuse and Rolling stone. Later, myotubes continue to 'grow' in a directed manner by this process of attraction, recognition and fusion, sending growth cone-like structures toward specific attachment sites. This analysis suggests that Hbs acts at the first step of this process, since free myoblasts in hbs mutant embryos do not cluster nearby muscles (like sns) (Bour, 2000). In addition, Hbs serves as a negative regulator of Sns activity. This negative regulation is necessary to complete the fusion process since unfused myoblasts are found in hbs mutant embryos (Artero, 2001).
There are several ways in which Hbs could antagonize Sns function at the molecular level. (1) Hbs could compete for a putative Sns 'ligand'. Given the similarity (69%) of the extracellular domains of Hbs and Sns, this appears reasonable. However, overexpression of the extracellular domain of Hbs alone can not block myoblast fusion, while such fusion is blocked by the full-length construct. Moreover, the data support a requirement for activity of the intracellular domain of Hbs. (2) Hbs and Sns could form a receptor complex that switches the positive Sns signal to a negative signal. (3) Another possibility would have Hbs and Sns function independently of one another but converge intracellularly on downstream proteins involved in fusion. For example, Echinoid, an Ig domain protein resembling Hbs in overall structure, has been shown to antagonize the Ras pathway in the eye, not at the cell membrane but intracellularly at the level of transcription of a target gene, tramtrack (Bai, 2001). Hbs, Sns and Kirre may also be functioning together similarly in the generation of the visceral muscles, since recent data indicate that visceral muscles are not mononucleated, as previously reported, but syncytial (Artero, 2001 and references therein).
Hbs and Sns co-localize at the cell membrane, which could correspond to focal adhesion points between founder cell and fusion-competent cells or among fusion competent myoblasts. However, Hbs precedes Sns temporally in the somatic and visceral mesoderm at stage 11, prior to the initiation of fusion. The two expression patterns largely overlap during stage 12-13 as fusion begins, with some Sns and Hbs-specific foci of expression, and by late stage 14, unfused myoblasts are basically devoid of Hbs but maintain Sns as fusion continues towards completion. These differences in the pattern of expression of Sns and Hbs are meaningful since when hbs is genetically removed such that fusion competent cells express Sns exclusively, a muscle fusion phenotype is detected. Hbs could, therefore, be revealing different potentials for the fusion-competent cells during muscle formation. Initially, cells that fail to become muscle progenitors during the first round of segregation may be included in a new equipotential cluster. These clusters appear largely to overlap and form in rapid sequence. Early Hbs expression could participate in maintaining myoblasts that fail to become progenitors in the first round of selection in an uncommitted state, ready for successive rounds of progenitor segregation. This provides a reasonable explanation for occasional muscle losses in hbs mutant embryos. Subsequently, upon adoption of fusion-competent cell fate and expression of Sns, Hbs could regulate early events in fusion, maintaining an orderly process. During later events in the fusion process (i.e. while myotubes are already growing towards their specific attachment sites), when there are fewer free fusion-competent cells, Hbs may no longer be necessary to regulate or orient fusion. Thus, three 'fusion-competent cell' stages are proposed (Artero, 2001):
Since founder cell markers such as Krüppel and Even-skipped define subsets of founders, it is envisaged that additional markers will be found that serve to characterize these different fusion-competent cell stages. Moreover, the hbs enhancer trap provides a novel tool to recognize subsets of fusion-competent cells without the technical difficulties associated with a punctate, membrane-bound signal (Artero, 2001).
Clearly, Sns is not the only potential partner for Hbs. Although Sns is expressed in the visceral mesoderm and muscle attachments, it is not expressed in heart, midline, hindgut, Malpighian tubules, imaginal discs or adult tissues where hbs expression and phenotypes are found. Either Hbs is acting alone in these tissues or it is interacting with other Ig domain proteins. Similarly, Sns does not always act with Hbs. Like Kirre (and unlike Hbs) Sns is expressed in garland cells (Artero, 2001).
Although Hbs operates in multiple places, one unifying theme to hbs pleiotrophy is found -- an association with Notch signaling. The evidence demonstrates that hbs is a novel target of Notch activity in the somatic musculature. There are also several compelling similarities between the hbs and Notch activities over the course of development. Weak gain-of-function Notch constructs block myoblast fusion, one of the aspects of hbs phenotype. Notch is involved in planar polarity in the eye and both hbs loss- and gain-of-function lead to polarity defects. Preliminary results suggest that the hbs semisterility phenotype is due to failure to specify stalk cells in the ovarioles, a phenotype described for Notch loss-of-function mutations. hbs overexpression in adults results in misplacement of bristles and hbs and Notch interact weakly in the wings, resulting in a measurable increase in wing nicks. Perhaps like the Enhancer of split complex, hbs could mediate part of Notch signaling. Future work will be directed to uncover the interaction between these two genes throughout development (Artero, 2001 and references therein).
With the identification of Sns and Hbs on fusion-competent myoblasts, the question arises as to what the corresponding extracellular partners may be on the founder cells/forming myotubes. IrreC-rst and Kirre are the Drosophila members of the DM-GRASP/BEN/SC1 subfamily of the IgSF. kirre is expressed by the founder cells but not the fusion-competent myoblasts (Ruiz-Gómez, 2000), and irreC-rst is expressed in the embryonic mesoderm but the identity of the cells was not specified. The return of kirre expression to the mesoderm can rescue the phenotype; however, rescue with irreC-rst was not attempted. So the respective contribution of the two proteins to the fusion phenotype is uncertain. However, since Kirre misexpression can guide myoblasts to novel locations, it is considered a founder cell-derived attractant (Dworak, 2001 and references therein).
The similar fusion phenotype for the sns mutant and the kirre/irreC-rst deletion and the attractive properties of Kirre suggest Sns and Kirre underscore a fusion-competent myoblast-founder cell attraction mechanism (Frasch, 2000). Meanwhile, hbs overexpression in the somatic mesoderm partially phenocopies the sns loss-of-function mutant and the irreC-rst/duf deletion, suggesting reduced attraction of myoblasts to the myotubes. However myoblast fusion is also partially blocked when IrreC-Rst is overexpressed in the mesoderm, and myoblasts go to ectopic locations when Kirre is presented in the epidermis. So the Hbs gain-of-function phenotype could also be interpreted as the response of myoblasts to an imbalance of attractive forces (Dworak, 2001).
Support for Hbs mediating an attractive function comes from the S2 cell assays. Under the given assaying conditions, neither Sns nor Hbs interacts homotypically, and Hbs does not bind to Sns in trans. These observations contradict a model where Hbs might block in trans an Sns-mediated attraction between fusion competent myoblasts and bias the interaction towards the Kirre-expressing founder cells. In the S2 cell aggregation assay, both Hbs and Sns show an interaction with Kirre, mediating heterophilic adhesion between S2 cells. But neither Sns nor Hbs induce aggregates in combination with IrreC-Rst or Side. These results support a model where both Hbs and Sns facilitate the Kirre-induced attraction of fusion competent myoblasts to founder cells. But the results do not rule out other interaction combinations between these different proteins. Further experiments are required to determine if they act in cis or in complexes, or whether they require different conditions for binding to one another in trans in the S2 cell assay (Dworak, 2001).
When hbs is globally expressed with the da-GAL4 driver, the myoblast fusion defect is enhanced, and muscle fiber insertions are also misplaced. To determine whether the latter is due to hbs misexpression in the musculature or the epidermis, hbs was misexpressed in the epidermis with several GAL4 drivers. When misexpressed in the epidermis within a hemisegment, subsets of muscles fail to traverse the hemisegment and either bunch ventrally (en-GAL4 and sca-GAL4 drivers) or align with the segment boundary (pnr-GAL4 drivers). As such, the misplaced muscle attachment phenotype observed in the da-GAL4 gain-of-function condition is attributed to epidermally rather than mesodermally misexpressed hbs (Dworak, 2001).
hbs is broadly expressed in the epidermis around and at the sites where muscles will ultimately attach, then becomes confined to the muscle attachment sites themselves. During normal development, Hbs may assist in slowing and constraining myotube exploration in the region where attachments must ultimately form. The data on myoblast fusion links Hbs to an attraction/adhesion mechanism. Furthermore, Kirre is present in developing mytotubes (Ruiz-Gómez, 2000) and Sns is also present at the muscle attachment sites (Bour, 2000). Given these expression patterns and the heterotypic interaction of Kirre with Hbs and Sns, it is possible that these proteins also interact during myotube guidance, serving to direct myotubes to their expidermal attachment sites (Dworak, 2001).
This is the first evidence of a direct physical interaction between extracellular molecules that are expressed on either fusion-competent myoblasts and at muscle attachment sites (Hbs and Sns), or on muscle founder cells and developing myotubes (Kirre). These observations suggest that adhesion between these molecules may aid recognition between fusion competent myoblasts and founder cells, and between myotubes and epidermal attachment sites. Whether the large cytoplasmic domains on these proteins have signaling abilities must now be determined (Dworak, 2001).
The hbs gene encodes protein domains that are characteristic of members of the immunoglobulin superfamily (IgSF). Translation of the sequence has uncovered a characteristic conserved start site followed by a stretch of hydrophobic amino acids typical of signal peptides. Following the signal sequence are six consecutive immunoglobulin-like (Ig) domains. The next region has the tryptophan and potential 'second' cysteine characteristic of Ig domains, yet lacks the first conserved cysteine. This modified Ig domain is followed by two complete Ig domains, then a single fibronectin type-III (FN) domain (Dworak, 2001).
There are 12 potential sites for asparagine-linked glycosylation on the ectodomain. Following the FN domain there are the 26 hydrophobic amino acids of the transmembrane domain, then a cytoplasmic tail consisting of 160 amino acids that contains 11 tyrosine residues. Analysis of the cytoplasmic tail with the PEST Sequence Utility program on the ExPASy Molecular Biology Server predicts a PEST sequence in the amino acids KSQSEAEPSNDDVYSK, starting at amino acid 1078 (Dworak, 2001).
The type, number and order of the Hbs extracellular domains are conserved in the Nephrin protein subfamily. Nephrins have been found in human (Kestila, 1998), mouse (Holzman, 1999), rat (Ahola, 1999) and C. elegans (Teichmann, 2000). Another Nephrin subfamily protein, Sticks and Stones (Sns) has been identified in Drosophila (Bour, 2000). The vertebrate Nephrin forms share close to 80% amino acid identity with one another. By comparison, Hbs shares 23% amino acid identity with the ectodomain of human Nephrin, 20% with the ectodomain of C. elegans Nephrin and 46% overall with Drosophila Sns. While the endodomain of the Nephrins is highly conserved between the vertebrate forms (80% identity between human and mouse), the fly forms differ greatly from the vertebrate forms (Hbs and vertebrates, 10% identity), the worm (Hbs and worm, 10% identity) and even each other (Hbs and Sns, 27% identity) (Dworak, 2001). In vertebrates, Nephrin is a component of the slit diaphragm, the molecular sieve that facilitates ultrafiltration in the kidney glomeruli (Somlo, 2000).
In a developmental Northern blot, a hbs transcript of 6.2 kb with high levels of expression was detected at mid-embryogenesis. hbs encodes a single pass transmembrane protein belonging to the Ig superfamily and contains nine Ig-C2 type repeats and a Fibronectin type III domain adjacent to its transmembrane region. A potential signal peptide sequence that is characteristic of membrane-bound proteins is present in the N terminus. BLAST database searches with Hbs reveal Nephrin to be the human ortholog (29% identity, 44% similarity). Mutations in Nephrin have been linked to the Congenital Nephrotic Syndrome of the Finnish type (Kestilä, 1998). Furthermore, there is a Drosophila paralog, the protein Sns, with 48% identity and 63% similarity to Hbs (Bour, 2000). The Hbs cytoplasmic domain, however, is shorter than its equivalent region in Sns (166 versus 376 amino acids, respectively). The Hbs extracellular region contains 13 NXS/T consensus triplets for N-glycosylation, eight of which are conserved in Sns. The cytoplasmic domain of the protein shows potential target sites for cAMP and cGMP-dependent protein kinases, protein kinase C (PKC), and casein kinase II (CKII). These phosphorylation sites are not conserved in Sns, except for a CKII site and a PKC site in two regions of strong conservation. In addition, Bour reported conservation of tyrosine residues between Sns and Nephrin in the intracellular domain, which are also present in Hbs. The Hbs cytoplasmic domain also contains an eight amino acid direct repeat, but no described conserved domains. Additional searches against the conceptual translation of predicted genes in the Drosophila genome with the intracellular region of Hbs did not find any significant match except with Sns (Artero, 2001).
date revised: 20 January 2001
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